Liquid crystalline phytosterol-glycerine complex for enhanced bioavailability and water dispersal

ABSTRACT

Edible phytosterol-containing compositions include molecular complexes of non-esterified phytosterols (P) and glycerine (G) in the form of liquid crystalline microparticles. Addition of an emulsifier (M) such as a monoglyceride or a modified lecithin, and optionally an ionic surfactant, to the complex facilitates its dispersal in an aqueous medium. A composition containing either the binary PG or ternary PGM molecular complexes can be formulated as a beverage, food product, or nutritional supplement. When administered to a human subject, the complexes sequester cholesterol in the gastrointestinal tract and reduce LDL cholesterol and total plasma cholesterol levels.

BACKGROUND

Cholesterol and phytosterols are very similar in molecular structure andare found in animal and plant cellular membranes, respectively. Bothchemical species serve as membrane structural elements and also servefunctional roles in living cells. These roles include affecting signaltransduction, protein and enzyme binding, membrane elasticity, and avariety of other functions. Cholesterol crystallization has beenimplicated in pathological conditions ranging from gallstone formationto arterial plaque and lesion formation. Maintaining cholesterol in asoluble or semi-soluble state, rather than a crystalline state, withinthe cell membrane is important. It remains unclear exactly how this isaccomplished within the complexity of a living cell's membrane; however,cholesterol combines with phosphatidylcholine, a phospholipid, whichseems to maintain cholesterol solubility. Nevertheless, there are limitsto the amount of cholesterol that can be combined with such aphospholipid, beyond which the cholesterol precipitates in crystallineform.

While preventing cholesterol crystallization has health implications andhas been the subject of a large number of research studies, theprevention of phytosterol crystallization has been less well studied,since the latter does not relate to a pathological state in humans.However, converting phytosterols from their inherently crystalline stateto a soluble or dispersed state, and in a micron-sized orsubmicron-sized microparticulate form, increases their biologicalefficacy, which is chiefly to facilitate fecal elimination ofcholesterol by admixing with cholesterol in the GI tract. To this end,phytosterols have been combined with a variety of edible solvents,co-solvents, emulsifiers and the like.

Phytosterols including beta-sitosterol, campesterol, stigmasterol andbrassicasterol are natural, edible, hydrophobic substances that arecommercially isolated from vegetable oils and tall oils. When ingested,these substances mix with dietary and endogenously synthesizedcholesterol, and can reduce the amount of cholesterol absorbed into thebloodstream to varying degrees. Like cholesterol, the phytosterolsreadily crystallize in a variety of morphologies (e.g., needles, platesand rods), all of which are poorly dispersible in water. Compositionsand methods have been described which are intended to increase theefficacy of phytosterols in eliminating cholesterol from thegastrointestinal tract. For example, emulsifiers have been used tofacilitate the dispersal of non-solubilized phytosterols. One suchsystem is described by Traska et al. in U.S. Pat. No. 6,423,363, whichdiscloses processed foods having an aqueous phase dispersion containinga high melting lipid, such as a phytosterol, that is emulsified with anon-sterol emulsifier. However, dispersions produced from phytosterolsand non-sterol emulsifiers that are melting together as described byTraska et al. and dispersed by shear in water typically containrelatively large microparticles (e.g., 10-15 microns). This substantialsize that can limit the bioavailability of phytosterols in binding andeliminating cholesterol in the GI tract, as well as the ability tomaintain stable suspensions in beverages and other useful compositions.The large size appears to be attributable to the crystalline structuremaintained in phytosterol-emulsifier mixed solids formed during coolingof molten mixtures described by Traska et al.

There remains a need to develop compositions that more fully and stablydisperse phytosterols in aqueous media for use in food and beveragecompositions.

SUMMARY OF THE INVENTION

The invention provides a binary intermolecular complex produced bycombining phytosterols that are highly hydrophobic, with glycerine, ahighly hydrophilic liquid. The complex is in the form of microparticleswith liquid crystalline structure. The complex is formed by heating andmelting free phytosterols together with glycerine, during which thephytosterol monohydrate becomes partially or substantially anhydrous. Incertain embodiments of the invention, an emulsifier such as amonoglyceride or a modified lecithin is added before cooling the binarycomplex, thereby forming a ternary or higher order complex that hasliquid crystalline structure and is highly dispersible in aqueous media.Forming these liquid crystalline complexes by melting the admixedingredients together renders the phytosterols water-dispersible for usein beverages, foods and dietary supplements.

The invention further provides ternary complexes containing glycerine,phytosterols, and one or more dispersing agents or emulsifiers, such asmonoglycerides (fatty acid monoesters of glycerine). The ternarycomplexes form microparticles with liquid crystalline structure that canbe efficiently dispersed in an aqueous environment. The microparticlescan be added to water-containing liquids (e.g., beverages and aqueousfoods), can be dispersed as remarkably small microparticles therein.

Thus, one aspect of the invention is an edible composition containing:(i) a non-esterified phytosterol, phytostanol, or a combination thereof(collectively “P”); and (ii) glycerine (“G”). In the composition P and Gare commingled to form, at least in part, a PG molecular complex, andthe weight ratio of G:P in the composition is at least 0.05:1. In someembodiments, the composition further contains a dispersing agent (“M”).In the composition, P, G, and M are commingled to form a PGM complex.The weight ratio of M:P is from about 0.1:1 to about 2:1, or in someembodiments from about 0.3:1 to about 1:1. In some embodiments, thecomposition further contains an ionic surfactant present at from 1% to10% by weight of the composition. In some embodiments, the compositionis in the form of spheroidal microparticles having a diameter in therange of about 1-2 microns or less. The microparticles containphytosterol-glycerine complexes organized at least in part into a liquidcrystalline structure.

Another aspect of the invention is a method of producing a compositioncontaining PG complexes. The method includes the steps of: (a) mixingone part by weight of P and at least about 0.05 parts by weight of G,and (b) heating the mixture, whereby a PG molecular complex is formed.Upon cooling, PG complexes are formed, which can be dispersed into anaqueous medium in the form of small microparticles having a diameter ofless than 5 microns, and preferably 1-2 microns or less. In someembodiments of the method, a dispersing agent M is admixed with theheated mixture containing P and G, and PGM complexes are formed. M isadded to about 0.1 to about 2 parts by weight, based on the weight of P.The addition of the dispersing agent improves the dispersibility inaqueous media of PGM complexes in the form of small microparticles ofless than 5 microns, and preferably 1-2 microns or less.

Another aspect of the invention is a beverage or food product containingthe PG or PGM complexes described above in the form of a suspension ofmicroparticles. The compositions of the invention are preferably ediblecompositions that are suitable for use as foods, beverages, or dietaryor nutritional supplements, or suitable for addition to foods, foodproducts, beverages, dietary or nutritional supplements for humans oranimals. Preferably the edible compositions contain only substances thatare recognized as foods, food additives, dietary supplements, orsubstances that are generally recognized as safe (GRAS) by the U.S. Foodand Drug Administration (FDA).

Yet another aspect of the invention is a method of using a beverages,food product, or nutritional supplement to treat or preventhypercholesterolemia. The method includes administration of thephytosterol- and glycerin-containing PG or PGM complexes described aboveto a subject in need of reducing their plasma cholesterol levels. Thecomplexes are administered in an amount effective to bind cholesterol inthe gastrointestinal tract and prevent or reduce its uptake, therebyreducing LDL and total plasma cholesterol (TC) levels in the subject. Insome embodiments of the method, the ratio of LDL to HDL cholesterol ofthe subject is also reduced.

The compositions and methods of the invention utilize glycerine toinhibit the commonly occurring crystallization of non-esterifiedphytosterols. The resulting complexes form microparticles that containliquid crystalline structure and are very small in size, such as in themicron range and submicron range. Their size is greatly reduced, atleast ten- to twenty-fold, compared to previous forms of phytosterols,and they have excellent dispersibility in water and aqueous media, suchas beverages, foods, and nutritional supplements. The microparticleshave a diameter of approximately 1-2 microns or smaller, offering athousand-fold decrease in individual microparticle mass compared toprevious forms of phytosterols, a dramatic increase in surface area-tovolume ratio, and a corresponding increase in bioavailability andefficacy for blocking cholesterol uptake in the gastrointestinal tract.Accordingly, glycerine complexes of phytosterols, with optional additionof emulsifier, greatly enhance the dispersal of phytosterols bypromoting the formation of small liquid crystalline microparticles, thatcan be stably dispersed in beverages and water-containing foods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a model of a binary complex (1:1 molar ratio) of aphytosterol and glycerine.

FIG. 2 shows a model of binary complexes of a phytosterol and glycerinewith alignment of the phytosterol molecules.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides new and advantageous compositions andmethods for improving the dispersal and bioavailability ofnon-esterified phytosterols for use in beverages, foods and dietarysupplements. The compositions include molecular complexes formed betweenphytosterol and glycerine molecules, with the optional addition of oneor more dispersing agents such as emulsifiers. The complexes formgenerally round-shaped, liquid crystalline microparticles of small size,in the micron and sub-micron range, which can be stably dispersed inaqueous media including foods, beverages, and nutritional supplementsthat can be administered to a human or animal subject to either reduceor enhance the uptake of cholesterol in the gastrointestinal system,depending on the phytosterol composition of the microparticles.

The phytosterols used in compositions of the invention can be any typeof non-esterified phytosterol. An intended use of the compositions is toreduce cholesterol uptake in a human or animal subject. As used herein,the term “phytosterol” refers collectively to both phytosterols andphytostanols. Phytosterols for use in the invention are preferablynon-esterified. Examples of suitable phytosterols includebeta-sitosterol, beta-sitostanol, campesterol, campestanol,stigmasterol, stigmastanol, brassicasterol, brassicastanol, clionasteroland clionastanol, and combinations thereof. Suitable phytosterols can bederived, for example, from vegetable oil, tall oil, or a combinationthereof. The phytosterols can be hydrated, hemi-hydrated, dehydrated, ora combination thereof.

The methods of the invention include combining, commingling, orcomplexing glycerine, which is an edible, polar, three-carbon polyol,with phytosterol to alter the crystallization and dispersal propertiesof the phytosterols and form new molecular complexes of phytosterol withglycerine. In these methods, the glycerine appears to function neitheras a solvent nor an emulsifier, but rather as a hydrogen-bonding,complex-forming agent that acts as a physical “spacer” molecule betweenneighboring phytosterol molecules. In addition to forming a new type ofmolecular complex, the addition of glycerine alters the physical andchemical associations among groups of phytosterol molecules, therebypreventing their aggregation or crystallization. This alteration isevidenced by a transformation from the crystalline state of the freephytosterol to the liquid crystalline state of the phytosterol-glycerinecomplex. The ability of glycerine, a hydrophilic molecule, to complexwith and at least partially separate phytosterol molecules is surprisingand unexpected. The formation of hydrogen bonds that link glycerine andphytosterols in the formed molecular complex may explain the more fluid,yet ordered, structure that characterizes the liquid crystallinephytosterol-glycerine complex.

While the phytosterol-glycerine complex of the invention can bedispersed in an aqueous environment, in certain embodiments anemulsifier such as a monoglyceride or a modified lecithin is added tothe phytosterol-glycerine binary complex to increase aqueous dispersalof the complex. As used herein, “free phytosterol” refers to uncomplexed(usually crystalline) phytosterol, “binary complex” refers to amolecular complex of phytosterol in association with glycerine, and“ternary complex” refers to phytosterol in association with glycerineand an emulsifier. The term “phytosterol complex” as used herein refersto the binary and/or ternary complex. Both the binary and ternarycomplexes may be present in the form of microparticles, such as asuspension of microparticles in an aqueous medium. An “aqueous medium”as used herein can be water or an aqueous solution or suspensioncontaining any desired solutes, such as salts, sugars, or chemicalssuitable for use in a food, beverage, or nutritional supplement, orcolloid particles such as micelles, proteins, aggregates, or fatdroplets.

Improved dispersal of the binary or ternary complexes is evidenced byformation of micron-sized and submicron-sized microparticles, which canbe useful to disperse the complexes in water, beverages, liquid formuladiets, and water-containing food products. The size (mean diameter) ofthe binary or ternary complexes can be, for example, less than 5microns, approximately 4 microns, approximately 2 microns, approximately1 micron, about 4 microns or less, about 2 microns or less, about 1micron or less, about 0.5 microns or less, or about 0.3 microns or less.In certain embodiments, the mean diameter of a population of PG or PGMmicroparticles can be about 1 to about 4 microns, about 2 to about 4microns, about 1 to about 2 microns, about 0.5 to about 1.0 microns,about 0.2 to about 0.5 microns, or about 0.1 to about 0.2 microns.Several known methods are available for determining the size of theindividual microparticles, or size distribution of a population of themicroparticles, including microscopy, light scattering, and sizeexclusion chromatography. The particles generally appear in the lightmicroscope as approximately spherical or spheroidal in shape. The smallsize of the microparticles is important for ensuring that they remainstably dispersed. The stably dispersed microparticles can remainsuspended in an aqueous medium for minutes, hours, or even days to weekswithout settling out or floating, depending on the properties of themedium, such as viscosity and specific gravity. Even after some settlinghas occurred, the microparticles can be readily re-dispersed byagitation of the suspension.

Either the binary or tertiary complexes can also be formulated asdietary supplements including pills, capsules or suspensions. Thepresence of emulsifier in such dietary supplements assures rapiddispersal of phytosterol complexes in the gastrointestinal (GI) tractafter ingestion of a composition containing the complexes. It isgenerally accepted that, for a given phytosterol preparation, smallerparticles that have greater surface area on a gram for gram basis aremore “bioavailable” and thus more clinically effective, than largerparticles for mixing with, binding, and eliminating cholesterol from theGI tract. The elimination of both dietary cholesterol, i.e., ingestedcholesterol, as well as endogenously synthesized cholesterol, results ina beneficial reduction in the level of undesirable plasma cholesterolsincluding LDL-cholesterol. It can be appreciated that various edibleingredients including mono- and diglycerides, lecithins, fats, and anynumber of other cooperative agents that assist in the binding,emulsification, and dispersal of phytosterols with cholesterol, may becombined in phytosterol formulations to further improve phytosterolbioavailability.

A method of making a phytosterol-glycerine binary complex according tothe invention includes combining glycerine with one or more phytosterolsto form a binary liquid crystalline complex. The method can includeheating, melting, and/or mixing in a blend the following components orcompositions comprising them:

(a) at least one non-esterified phytosterol and/or phytostanol(abbreviated “P”); and

(b) glycerine (abbreviated “G”).

Binary complexes of this type are abbreviated PG. Optionally, propyleneglycol, another edible three-carbon hydrophilic liquid, also may beincluded in the blend, and can substitute for all or part of theglycerine. According to the method, the P and G components are mixed orcommingled, preferably in a melted or liquid state, using any desiredmixing equipment, such as conventional mixers used in the food orchemical industry, blenders, propellers, homogenizers, etc. The step ofmixing or commingling should provide sufficient mixing action and becarried out for sufficient time to permit molecular complexes to beformed between the phytosterol and the glycerine. The step of mixing orcommingling can be carried out at a sufficiently high temperature (e.g.,70° C. or more, or 100° C. or more) to maintain the phytosterol in amelted (liquid) state but also to encourage any water of hydration todissociate from the phytosterol component and be removed by evaporationor boiling. The final weight ratio of the mixed components G:P is atleast 0.5 to 1 (0.5:1). Preferably, enough glycerin is added so that allof the phytosterol in the composition is complexed as a PG complexhaving an approximately 1:1 molar ratio of glycerine to phytosterol. Insome embodiments, an excess of glycerine is added, such that the molarratio of glycerine to phytosterol is greater than about 1:1.

In another method according to the invention, a ternaryphytosterol-glycerine-emulsifier complex is prepared by admixing andheating the above binary blend (i.e., (a)+(b)), either at the same timeas the phytosterol and glycerine are combined, or subsequently, with thefollowing dispersing agent component or a composition comprising it:

(c) at least one dispersing agent (abbreviated “M”), such as amonoglyceride (e.g., glyceryl monopalmitate or glyceryl monostearate), adiacylglyceride, a lecithin such as a modified lecithin (e.g.,hydrolyzed sunflower lecithin), an ionic surfactant, or a combinationthereof. As used herein, a “dispersing agent” is a chemical agent, suchas emulsifier or surfactant, that increases the dispersal of phytosterolcomplexes in an aqueous medium above the level that occurs in theabsence of the dispersing agent. Preferred dispersing agents areemulsifiers. Dispersing agent M preferably is added to the mixture of Gand P to give a final amount of about 0.1 to about 2.0 parts by weightbased on the weight of P. Addition of a dispersing agent to PG complexesforms ternary complexes (abbreviated “PGM”). Examples of suitabledispersing agents include monoglycerides (e.g., glyceryl monopalmitate,glyceryl monostearate, and combinations thereof), lecithins (e.g.,hydrolyzed sunflower lecithin, or another hydrolyzed or hydroxylatedlecithin), and triglyceride-based oils or fats. In certain embodiments anon-ionic emulsifier is combined with from about 1% to about 10% byweight (based on the total weight of M) of an ionic surfactant. Examplesof suitable ionic surfactants include a salt of a fatty acid, whereinthe fatty acid is selected from the group consisting of stearic acid,palmitic acid, myristic acid, lauric acid, capric acid, caprylic acid,oleic acid, and combinations thereof. A preferred dispersing agent isthe combination of one or more monoglycerides with 5 weight % of sodiumstearate. Another preferred dispersing agent contains a modified (e.g.,hydrolyzed) lecithin and an ionic surfactant, such as sodium stearate.Ternary PGM complexes are generally more highly dispersible in anaqueous medium than corresponding binary PG complexes.

Both binary and ternary complexes possess a liquid crystalline structureand a very small microparticle size (typically 1-2 microns or less) inaqueous dispersions. Such dispersions differ markedly in their dispersalproperties from those prepared with more conventionally crystallizedmicroparticles (typically 10-50 microns) obtained by a process ofmelting and co-crystallizing phytosterols and monoglycerides in theabsence of glycerine.

In addition to being supplied as an aqueous dispersion, the phytosterolcomplexes can be supplied in the form of a paste, as granules, or as apowder, any of which can be added to a food, food item or food product,liquid food additive, beverage, nutritional beverage, or nutritional ordietary supplement either during formulation of a commercial product orby the end user. Examples of suitable nutritional beverages includecow's milk, sheep's milk, goat's milk, soymilk, almond milk, and coconutmilk Examples of suitable food items include yogurt, cottage cheese,sour cream, soup, salad dressing, tomato catsup, mustard, barbecuesauce, steak sauce, Worcestershire sauce, cocktail sauce, tartar sauce,pickle relish, tomato-based pasta sauce, pizza sauce, prepared chili,and dessert sauce.

The very small average diameter of PG and PGM liquid crystallinemicroparticles, such as PGM microparticles formed with monoglyceridesand/or lecithins, allows these microparticles to remain dispersed inbeverages or liquid foods almost indefinitely without settling, whilehelping to maximize their bioavailability for binding cholesterol. Theuse of a monoglyceride in forming a ternary PGM complex is preferred,because on a gram-for-gram basis monoglycerides provide more efficientdispersal over the use of fat (i.e., triglycerides) for dispersingphytosterols. While fat has been effectively used for phytosteroldispersal (e.g., forming a “TRP” complex as described in U.S. Pat. No.7,144,595), human digestion of fat, i.e., triglyceride molecules, yieldssn-2 monoglycerides by the action of pancreatic lipase enzymes in the GItract. Accordingly, monoglycerides are actually much more effective on aweight basis as dispersing agents for phytosterols than fat. Further,supplying one part by weight of monoglycerides in the presentlydescribed PGM complex is expected to provide the equivalent dispersingactivity (with only ⅓ the calories) of up to a three parts by weight offat.

A useful PGM complex may be formulated, for example, by heating andmelting together approximately 1 part by weight non-esterifiedphytosterols with 0.5 part by weight glycerine and approximately 0.5 to2 parts by weight (e.g., 1 part by weight) of a monoglycerideemulsifier, such as Myvatex 8-60 manufactured by Kerry Ingredients andFlavours, Beloit, Wis. The latter contains glyceryl monostearate,glyceryl monopalmitate and a small amount (i.e., 4-6% by weight) ofsodium stearate. This exemplary PGM complex formula containsapproximately 40% by weight of phytosterols. Formulations containinghigher or lower proportions of glycerine and emulsifier relative tophytosterols may also be constituted. The melting temperatures oftypical PGM complexes tend to be conveniently reduced (e.g., 60-90° C.)when compared with pure phytosterols (e.g., 135-140° C.) owing to thepresence of lower melting point emulsifiers such as a fatty acidmonoglycerides.

In summary, a highly dispersible liquid crystalline PGM complex can bemade by heating and melting together non-esterified phytosterols,glycerine, and an emulsifier such as a monoglyceride, a lecithin, oranother dispersing agent. Such PGM complexes are up to 100% dispersiblein aqueous media and can be incorporated into beverages and foods byblending, used to make a liquid concentrate for addition to such foodsand beverages, or formulated as dietary supplements, including pills,capsules, and liquid dietary supplements. The resulting liquidcrystalline PGM dispersions contain microparticles of a small size inthe micron to sub-micron range, which is believed to represent thesmallest particle size distribution of any edible phytosterolcomposition reported prior to the invention.

The Phytosterol-Glycerine Complex

Glycerine (also known as glycerin, glycerol, or C₃H₈O₃) is a relativelylow molecular weight (MW=92) water-soluble, polar compound which isliquid at room temperature and has low vapor pressure. It is acolorless, odorless, edible, sweet-tasting hygroscopic liquid that iswidely used in pharmaceutical preparations and foods. Glycerine isgenerally recognized as safe (GRAS) for use in foods, and is categorizedby the FDA and the American Dietetic Association as a carbohydratesweetener. It is produced by many companies as a by-product of makingsoap, biodiesel fuels and refining edible fats and oils. Its threehydroxyl groups are responsible for its water-miscibility andhygroscopic nature. Glycerine is a precursor for synthesis oftriglycerides and of phospholipids in the liver and adipose tissue. Whenthe body uses stored fat as a source of energy, glycerine and fattyacids are released into the bloodstream. Nutritionally, glycerine is acarbohydrate that can be enzymatically converted into glucose by theliver to provide energy for cellular metabolism. Before glycerine canenter the pathway of glycolysis or gluconeogenesis, it must be convertedenzymatically to the intermediate, glyceraldehyde 3-phosphate. Glycerineis known to protect lipid membranes in cells, and may prevent damage dueto osmotic stress and dehydration. Glycerine is also well known as amoisturizer or humectant for human skin. In view of these properties, itwas surprising to find that glycerine can form an intimate complex withthe hydrophobic and usually crystalline phytosterols.

The invention provides an associative chemical complex formed bycombining glycerine, an edible liquid, with melted phytosterols andcommingling the melted mixture to form the complex. This complex isbelieved to result from intermolecular hydrogen bonding of one or moreof the hydroxyl groups found in glycerine with the single hydroxyl groupfound in the phytosterol molecule. The complex fails to crystallize astraditional phytosterol crystals (e.g., needles, rods, or plates);instead, the complex disperses the phytosterol material in aqueous media(e.g., water, beverages, water-based foods, controlled nutritionalbeverages, dietary supplements, saline, cell culture media, oraquaculture media). Formation of the readily dispersed PG or PGMcomplexes also facilitates chemical mixing and association ofphytosterol molecules with cholesterol molecules in the GI tractfollowing phytosterol ingestion.

Non-esterified phytosterol (i.e., free phytosterol) preparations usedherein are purified, food grade materials typically containing in excessof 90% free phytosterols. The phytosterol compositions may includevarying proportions of beta-sitosterol, campesterol, stigmasterol andbrassicasterol as well as reduced or hydrogenated chemical forms knownas stanols. Suitable commercial sources for free phytosterols includeVegapure® FS (Cognis Corp., La Grange, Ill.), CardioAid™ non-esterifiedphytosterols from soybeans (Archer Daniels Midland, Inc., Decatur, Ill.,also known as ADM, Inc.) and CoroWise® FG-50 (Cargill, Inc.,Minneapolis, Minn.).

Non-esterified phytosterols routinely exist as monohydrated moleculesbut can also exist as hemi-hydrated and anhydrous forms depending uponthe temperature and the surrounding chemical environment. Upon heatingin a fat or in glycerine to a temperature above the boiling point ofwater, a suspension of crystalline phytosterol powder is observed toboil briefly as the phytosterol monohydrate becomes anhydrous and watermolecules are evolved as steam. With such heating in the presence ofglycerine, it is believed that a glycerine molecule replaces the watermolecule previously hydrogen-bonded to the hydroxyl moiety of thephytosterol molecule, with a hydrogen bonding interaction between ahydroxyl group on glycerine and the phytosterol hydroxyl. In theprocess, the phytosterol becomes either fully or partially dehydrated.Upon cooling, the glycerine-sterol complex forms amorphous (i.e., not aregular crystalline solid) micro-spherules that further examination hasshown to contain liquid crystalline material rather than conventionalrigid crystals. The glycerine-complexed phytosterol molecules stillcohere with one another about as strongly as the original phytosterolmonohydrate molecules, evidenced by maintenance of an elevated meltingpoint (in excess of 130° C.). However, as described below, the presenceof glycerine in the liquid crystalline phytosterol binary complexstructure pre-disposes this structure to enhanced disruption by waterafter emulsifiers have also been incorporated into the structure to forma ternary complex structure.

It was discovered that a substantial amount of glycerine can be combinedwith phytosterol to form a complex (up to approximately 20% glycerinebased on the weight of phytosterol). This was surprising given thatglycerine is a low molecular weight hydrophilic substance whereasphytosterols (e.g., beta-sitosterol, campesterol and stigmasterol) arehighly hydrophobic substances. It was even more surprising given thatwhen a small amount of phytosterol, e.g., 1% by weight, is heated inglycerine to a temperature that exceeded the melting temperature of thephytosterol (e.g., 150° C.), only a negligible amount of the phytosteroldissolves in the glycerine. The question arose, why can 20% glycerinedissolve into melted phytosterols when a small amount of phytosterolwill not dissolve into glycerine. It was also difficult to explain why,when glycerine was replaced with propylene glycol, another smallhydrophilic liquid, the propylene glycol and phytosterols were found tobe substantially miscible at 150° C. For example, equal weightpercentages of these ingredients dissolve easily in one another.

The approximate 20% by weight saturation level of glycerine relative tophytosterols represents the formation of a 1:1 molecular stoichiometriccomplex in which one glycerine molecule forms a hydrogen bond at thehydroxyl group of one phytosterol molecule (glycerine/sterol molecularweights=92/415=22%). This phenomenon differs from chemical solubility inwhich a solute and solvent are not constrained by formation of such acomplex. The amount of glycerine present in such a complex isinsufficient for the glycerine to form a bulk liquid phase, and in anyevent phytosterols are too hydrophobic to dissolve in a polar liquidsuch as glycerine.

The above-described melted mixtures of phytosterols and glycerine orpropylene glycol were cooled to form a solid mass and then investigatedby phase contrast light microscopy. With propylene glycol, thephytosterols solidified principally as crystalline particles ofapproximately 20-200 microns, whereas the glycerine-associatedphytosterols solidified as masses of countless microparticles, eachmeasuring approximately 1-2 microns or less in diameter and spherical orspheroidal in shape. The microspheres appeared amorphous (i.e., not aregular crystalline solid) as viewed using phase contrast illumination.With regard to dispersibility, the glycerine-complexed material was farsuperior to the propylene glycol material.

Polarized light microscopy allows determination of the extent ofordering and alignment of molecules within PG or PGM microparticles.While the overall shape of the microparticles appears round andnon-crystalline, their appearance under polarized light confirms thatthey contained highly oriented molecules characteristic of liquidcrystals. Thus, when the above-described glycerine-containingphytosterol samples were placed on a microscope slide and examined at100×-200× magnification under transmitted polarized light (Olympus ModelBX-51 microscope) essentially 100% of the microparticles appearedalternately bright (polarized light-transmitting) and subsequently dark(polarized light-non-transmitting) as samples were progressively rotatedon the microscope stage. Therefore, glycerine-complexed phytosterolmolecules maintain a physical and optical alignment in spite of the factthat solid phase crystals characteristic of simple phytosterols were notformed during cooling and solidification of the complex. Interestingly,while an elevated temperature allowed the hydrogen-bondedglycerine-sterol material to melt, and to separate into micron-sizedmicrospheres, the phytosterol molecules in the cooled microspheres werehighly ordered.

Phase contrast microscopy allows differentiation among particulatestructures based upon their external morphology. “Crystals” areidentified by their angular appearance (e.g., angular-shaped polygons,plates, rods and needles) and their light refraction. This contrastswith the appearance of “amorphous” or “non-crystalline” particles, whichare typically round or globular in shape and appear either dark orexhibiting reduced light refraction. On the other hand, a “mesomorphic”or “liquid crystalline” state, as used herein, refers to a physicalstate of matter (including multiple intermediate transition states) thatis intermediate between a liquid and a fully solid state. The solidstate, in the case of substantially pure phytosterols, is typicallycrystalline. Thus, substances and microparticles formed from suchsubstances, that originally exist in a crystalline state may, because ofan alteration in chemical make-up (e.g., combining with glycerine)undergo a transition through one or more intermediate so-called liquidcrystalline or mesophase states. During this transition, mesomorphicsubstances may melt and begin to flow, assuming rounded shapescharacteristic of liquids rather than solids, even though the substancesmay retain some crystalline properties. For the purposes herein, suchmesophases or liquid crystalline states for phytosterol-containingparticles and microparticles are termed “amorphous” or “liquid”materials (as opposed to solid or crystalline materials) provided thatthey are fluid. Fluidity is shown for these materials when they formgenerally round microparticles rather than optically refracting angularsolid particles. Such fluidity is functionally important because itallows formation of very small microparticles and thus large surfaceareas that favor increased phytosterol bioavailability.

The addition of glycerine to phytosterol, and the consequent formationof a phytosterol-glycerine complex, is very useful because the complexforms very small microparticles whose ratio of surface area to mass isvery great. The tendency of these microparticles to aggregate can beopposed either by mixing to apply a shear force using conventional dairyhomogenization equipment, high shear blade blending, propeller mixersand the like, or through the addition of emulsifiers. Addition of anemulsifier to the phytosterol-glycerine melt causes the microparticlesto disaggregate into individual microparticles. Since the microparticlesoffer a vary small diameter and maximized surface area, they producedispersions that remain stably suspended in aqueous media, such asbeverages, for long periods of time without settling. A furtheradvantage of the microparticulate form of PG and PGM complexes is theirhigh degree of bioavailability.

Hydrogen Bonding in Phytosterol-Glycerine Complexes

The chemical interaction between phytosterol molecules and glycerine inPG and PGM complexes of the invention are believed to involve a 1:1molecular complex formed by hydrogen bonding between glycerine andphytosterol molecules. The expected molecular associations are showndiagrammatically in FIGS. 1 and 2, depicting beta-sitosterol andglycerine molecules. These complexed molecules can form orderedstructures in which phytosterol molecules align parallel to one another.In a hydrophobic environment, the pairwise complex shown in FIG. 1 maybe favored, since this arrangement locates the hydrophilic glycerinemolecules oriented inward. Thus, in a heated anhydrous mixturecontaining mobile beta-sitosterol and glycerine molecules, thehydrophobic phytosterol molecules are expected to self-associate, andthe glycerine molecules should likewise self-associate as their freehydroxyl groups form hydrogen bonds with one another. By contrast, in anaqueous environment, most glycerine molecules should be oriented outwardto maximize hydrogen bonding with water (FIG. 2). While not intending tolimit the invention to any particular structure of the phytosterolcomplexes or molecular bonding arrangement, it is believed that theplanar phytosterol ring structures are stacked into parallel arrays asindicated in FIG. 2.

Hydrogen bonding between glycerine and the hydroxyl group of thephytosterol molecule weakens or disrupts the hydrophobic interactionsthat give rise to normal crystallization following melting and coolingof either a single molecular species (e.g., beta-sitosterol) or mixedspecies (e.g., soybean oil-derived phytosterols). This results in theformation of a non-crystalline solid when a molten mixture of glycerineand phytosterols is cooled to room temperature. While not intending tolimit the invention to any particular mechanism or molecular structure,it is believed that two glycerine molecules forming a double bridgebetween two neighboring beta-sitosterol molecules would produce a 1:1stoichiometric molecular complex, as depicted in FIG. 1. Based on theirmolecular weights, 92 g of glycerine would combine with 415 gbeta-sitosterol. This would correspond to a weight ratio of 22 partsglycerine to 100 parts phytosterol, and it is consistent with theobservation that somewhat more than 20 parts by weight of glycerine areable to dissolve during mixing with molten phytosterols. Between 20 and30 parts by weight were observed to prevent crystallization of 100 partsby weight of phytosterols.

With regard to the third hydroxyl group in the glycerine molecule thatis not believed to participate in the hydrogen bonding structure shownin FIG. 1, it would be available for hydrogen bonding with a polarmolecule, e.g., water, or an emulsifier. This can occur when otheringredients are mixed together with the binary phytosterol complex in afood or beverage composition. By contrast, as shown in FIG. 2, in anaqueous environment two of the three glycerine hydroxyl groups areavailable for assisting in dispersal of the phytosterol molecule. Thus,the glycerine molecule, when hydrogen bonded to a phytosterol molecule,may increase the density of chemically available hydroxyl groups fromone (the original phytosterol hydroxyl group) to at least two.

It is interesting to compare and contrast the role of glycerine with therole of an emulsifier in terms of chemical interaction with phytosterolmolecules. An emulsifier molecule must generally be of sufficient sizeto be amphiphilic. That is, the emulsifier molecule should contain atleast one “water-associating” or hydrophilic portion, and at least one“fat-associating” or hydrophobic portion. These two different portionsallow the combining of liquids that normally do not mix, such as fat andwater. Thus, lecithin from egg yolk is amphiphilic and can stabilize fatmicrodroplets suspended, for example, within a continuous “external”phase of aqueous vinegar and other flavorings to create the emulsionrecognized as mayonnaise. While varying amounts of emulsifiers may beadded for stabilizing such emulsions, there are typically no “solubilitylimits” per se with the use of an emulsifier because the emulsifieroccupies a separate interface position in a liquid system between twocomponents that do not dissolve in one another, e.g., oil and water. Bycomparison, glycerine is a small three carbon-containing molecule thatis either immiscible or miscible to varying degrees in other solvents.By conventional definitions, glycerine is a solvent or co-solvent ratherthan an emulsifier. When mixed with phytosterols that have been meltedat a temperature of approximately 135° C., glycerine reaches whatappears to be a solubility limit (i.e., saturation level) at about 25 gper 100 g phytosterols. Such solubility limits are commonly encounteredamong solvents and co-solvents. For example, alcohol molecules whosemolecular structures contains more than three carbon atoms reach definedsolubility limits in water, e.g., n-butanol @ 9.1 ml per 100 ml water,n-amyl alcohol @ 2.7 g per 100 g water. However, in the presentinvention glycerine acts to alter the crystal form of phytosterolsrather than as a solvent or as an emulsifying agent.

Dispersing Agents

The binary complex formed between glycerine and phytosterols can bemodified to form a more highly water-dispersible ternary complex byfurther adding a dispersing agent to the heated blend of meltedphytosterol and glycerine. A variety of emulsifiers have been shown tobe capable of dispersing the phytosterol-glycerine complexes in water orother aqueous media. For example, monoglycerides such as glycerylmonostearate or glyceryl monopalmitate, or modified lecithins can beused as the dispersing agent. Monoglycerides are preferred over otheremulsifiers because they contain fatty acids that enhance thebioavailability of non-esterified phytosterols (see Perlman et al., U.S.Pat. No. 6,638,547). Since fat molecules (triglycerides) are convertedby lipase enzymes to monoglycerides during digestion, it is likely thatmonoglycerides also act as the biologically active dispersing agent whenfats are used as a phytosterol carrier vehicle and combined with foods.

Ternary molecular complexes containing monoglycerides commingled with PGcomplexes can be easily produced by admixing and melting all of theingredients (sterol, glycerine, and emulsifier) together and coolingthem. Alternatively, the ternary blend can be made in successive stepsby first making a PG complex and then admixing the monoglyceride. Theresult is a plastic-like ternary solid, which like the binary PG solid,appears amorphous rather than crystalline when examined by phasecontrast microscopy. However, polarized light microscopy shows that theternary complex, like the binary complex, forms a liquid crystallinemolecular structure. The preponderance of liquid crystalline materialwas observed by rotating specimens of these complexed materials on glassslides supported on the microscope stage during transmission ofcross-polarized light. As the specimen is rotated, localized portions ofthe material (including individual micro-spherules) visually transitionbetween bright and dark as the polarized light is alternatelytransmitted and not transmitted through the liquid crystalline material.By direct visual examination, the liquid crystalline state ofglycerine-containing phytosterol material supports formation ofultra-small fluid microparticles. The complexes are fully and relativelyeasily dispersible in water and other aqueous liquids (e.g., cows milkand soymilk) as well as aqueous foods, in the form of tiny micron andsub-micron-sized spherules (≦2 μm or ≦1 μm in diameter).

The presence of glycerine in a binary complex blocks the crystallizationof phytosterol, and in a ternary complex with phytosterol and adispersing agent such as a monoglyceride blocks co-crystallization thatotherwise occurs when these constituents are melted together and cooled.When unaltered by glycerine, the latter binary complex of phytosterolsand monoglyceride (a PM complex) crystallizes quickly, as can be viewedby phase contrast microscopy, and produces a less stable and less usefulsuspension of larger crystalline microparticles in water (20-100 micronsin diameter). Such crystals have poor bioavailability. Smaller fluid(non-crystalline) microparticles have the advantage of providing a muchgreater surface area for a given amount of material than largerparticles. Therefore, the smaller amorphous glycerine-containing ternaryphytosterol microparticles will have superior bioavailability forcombining with and binding cholesterol than the larger binarycrystalline PM particles formed without glycerine. With the advantage ofgreater cholesterol binding, the smaller phytosterol-containingmicroparticles formed with the benefit of glycerine are expected toincrease fecal elimination of cholesterol from the GI tract and therebyfurther decrease mammalian plasma LDL cholesterol levels.

In addition to inhibiting the crystallization of phytosterols, glycerineappears to modify the chemistry of monoglyceride emulsifiers and theirformation of complexes with phytosterols. The interaction betweenglycerine and monoglycerides may beneficially inhibit the formation oflarger binary crystals that are otherwise formed when monoglyceridessuch as glyceryl monostearate co-crystallize with phytosterols. Instead,an amorphous and physically plastic ternary mixture is formed, thatincludes glycerine, phytosterols (including beta-sitosterol for example)and at least one amphiphilic dispersing agent or emulsifier, such as amonoglyceride (e.g., glyceryl monostearate). The dispersing agentfacilitates dispersal of the ternary mixture as microparticulatespherules in any aqueous medium. Upon dispersal in water, for example,the spherules containing glycerine-modified phytosterols and emulsifierappear to be smaller than other commercially available phytosterolparticles. The average diameter of these microparticles is many-foldsmaller than crystalline microparticles formed without the benefit ofglycerine. As a result of their smaller size, PGM microparticles (e.g.,containing glycerine-phytosterol-monoglyceride) are expected to haveimproved bioavailability when ingested, compared to either unmodified PMparticles (e.g., phytosterol-monoglyceride) that share the samecombination of phytosterols and monoglyceride emulsifiers but areformulated without the benefit of glycerine. Accordingly, PGMmicroparticles are expected to bind increased levels of cholesterol inthe digestive system, promoting greater fecal elimination ofcholesterol, thereby further reducing plasma LDL cholesterol levels.

Emulsifiers are amphiphilic agents that enable the dispersal ofphytosterol-glycerine complexes in aqueous media and a diversity ofwater-containing edible materials. Examples of suitable emulsifiersinclude edible non-ionic and ionic surfactants, e.g., mono- anddiglycerides, unmodified and modified lecithins (e.g., hydrolyzed andhydroxylated lecithins), and synthetic emulsifiers such as acetic,lactic, citric, and succinic acid esters of monoglycerides, diacetyltartaric acid ester of mono- and diglycerides (DATEM), polyglycerolesters of fatty acids, sorbitan esters of fatty acids and sucrose estersof fatty acids, edible salts of fatty acids, and combinations of these.One example of a useful monoglyceride emulsifier is a combination ofglyceryl monostearate and glyceryl monopalmitate derived from palm oil;this is available as Myvatex 8-60, manufactured by Kerry Ingredients andFlavours (Beloit, Wis.).

An unmodified lecithin that has a low solubility in water may becombined with a modified lecithin or other emulsifier(s) to form a mixedamphiphilic emulsifier. Lecithins used herein are preferably modifiedsuch that they are more hydrophilic relative to unmodified lecithins. Insome embodiments, a natural vegetable lecithin is modified by eitherhydroxylation or hydrolysis (e.g., modified sunflower lecithin),rendering the lecithin sufficiently hydrophilic so that when combinedwith a preformed PG complex, the resultant amphiphilic particles aredispersible in water-containing liquids (e.g., cow's milk or soymilk).

When considering emulsifiers, it may be useful to consider theirhydrophilic-lipophilic balance (HLB). The HLB value may be calculatedbased on values for the different regions of the emulsifier molecule. W.C. Griffin's method for classifying non-ionic emulsifiers by their HLBvalue (J. Soc. Cosmetic Chemists 1:311 (1949)) considered the molecularmass of the hydrophilic portion of a molecule compared to the wholemolecule, to provide an HLB number on an arbitrary scale of 0 to 20. Avalue of 0 corresponds to a fully lipophilic molecule while a value of20 corresponds to a fully hydrophilic molecule. According to Griffin,the HLB value predicts the surfactant properties of a molecule. Morespecifically, a value from 4 to 6 indicates a water in oil (w/o)emulsifier while a value from 8 to 18 indicates an oil in water (o/w)emulsifier. In certain embodiments of the invention, emulsifiers areused that emulsify the glycerine-sterol complex into water. In someembodiments, the applicable HLB range is about 8 to about 18. Inparticular embodiments, lecithins used in the amphiphilic emulsifiersdescribed herein can be modified such that they have an HLB range ofabout 8 to about 18. In other embodiments, an amphiphilic emulsifier ormixture of amphiphilic emulsifier molecules having both lipophilic andhydrophilic chemical properties can be used. In yet other embodiments,because beverages to be supplemented with the glycerine-sterol complexinclude cow's milk and soymilk that are often purchased byhealth-conscious consumers, the emulsifier can be derived from a naturalsource. For example, lecithin that is prepared directly or indirectlyfrom a natural food source material can be used. In certain embodiments,the emulsifier may include chemically synthesized emulsifiers, such as asorbitan derivative or a polyethylene glycol.

In one embodiment, the amphiphilic emulsifier includes hydrolyzedsunflower lecithin (Giralec® HE-60 or Giralec® H-US produced byAustrade, Inc., Palm Beach Gardens, Fla.). In some embodiments, fromabout 90% to about 99% by weight of a preformed phytosterol-glycerinecomplex is blended with from about 10% to about 1% by weight of amodified lecithin to produce microparticles dispersible in liquids suchas beverages and fluid foods. In other embodiments, from about 94% toabout 98% by weight of the preformed phytosterol-glycerine complex isblended with from about 8% to about 2% by weight of modified lecithin.

In certain embodiments of dispersing agents, lecithin, or another lipidsuch as a diacyl glycerol or triacylglycerol, is hydrolyzed usingenzymatic phospholipase A rather than acid or base hydrolysis, allowingthe beta (sn-2) fatty acid to be selectively removed. In otherembodiments, hydroxylation of lecithin or another lipid is performed byreacting lecithin with hydrogen peroxide and lactic or acetic acid. Inparticular embodiments, hydroxyl groups are added at sites ofunsaturation in the lecithin's fatty acids.

Modified lecithins used as dispersants in the present invention include:Yelkin®1018 soy lecithin (hydroxylated) with an HLB of 9 (ADM, Inc.);Alcolec® C LPC20 canola lecithin (enzyme-hydrolyzed) with an HLB of 12(American Lecithin Company (ALC, Inc.), Oxford, Conn.); Alcolec® EM soylecithin (enzyme-hydrolyzed) with an HLB of 9 (ALC); and Giralec®HE-60sunflower lecithin (enzyme-hydrolyzed) with an HLB of 8-9 (Austrade,Inc., Palm Beach Gardens, Fla.). In certain embodiments, modifiedlecithins certified as produced from natural non-genetically modifiedorganisms can be used.

Sterol-glycerine complexes were found to be uniformly and stablydispersed throughout a liquid aqueous medium using modified (e.g.,hydrolyzed or hydroxylated) lecithins having HLB values of between about8 and about 12 that are typical for emulsifiers of oil in water. Natural(unmodified) lecithins may not be sufficiently active in some cases toachieve the desired uniform and stable dispersal. As used herein, stabledispersal of PG or PGM microparticles means that the particles do notseparate (float or sink) from the liquid to which they are added, thatis, to the extent that they can't be re-dispersed with shaking.

As utilized and defined in the presently described formulations,monoglycerides are dispersing agents or emulsifiers, but glycerine (orpropylene glycol) is not a dispersing agent or emulsifier. Thisdifferentiation is based on chemical and molecular affinities, and thefact that the monoglyceride molecule includes both a hydrophobic fattyacid moiety and two hydrophilic hydroxyl groups that enable thisamphiphilic emulsifier molecule to bind and combine with bothhydrophobic phytosterol molecules and water. Glycerine, however,contains three hydroxyl groups and is miscible with water, and isbelieved to interact with phytosterols through hydrogen bonding asexplained above. Accordingly, glycerine appears to act neither as anemulsifier or dispersing agent nor as a solvent in forming a complexwith phytosterols.

In addition to forming a complex when combined with melted phytosterols,glycerine exhibits partial solubility in melted monoglycerides. Thus forexample, combining (in the absence of water) 10% by weight glycerinewith the commercial Myvatex 8-60 monoglyceride product, results in asignificant decrease (6-8° C.) in the 70° C. melting point of themonoglyceride. This melting point depression is consistent with thephysical chemistry of a crystalline solid whose structure is interruptedby a solute molecule. In this case, glycerine, as a solute molecule mayinteract with a monoglyceride, thereby decreasing the monoglyceridemelting point. Larger amounts of glycerine (e.g., 30-50% or more byweight) combined with the same monoglycerides induce a different change,i.e., the prevention of crystallization and promotion of gel formation.

Co-crystallizing complexes of phytosterols and monoglycerides isdescribed by Akashe, et al. in U.S. Pat. No. 6,267,963. Such complexesformed between emulsifiers and phytosterols have substantially lowermelting temperatures than phytosterols alone (see Example 2 in U.S. Pat.No. 6,267,963). By contrast, when a PG complex containing a 1:1 weightratio of glycerine (with or without propylene glycol) and phytosterols(soybean or tall oil-derived phytosterols) is heated, melted and cooledto room temperature, the re-melting temperature (128-130° C.) is onlyslightly lower than that of the original phytosterols (approximately132-134° C.). This finding suggests that the chemical complex formedbetween glycerine and phytosterol may be relatively weak. Thus,glycerine appears to interact uniquely with non-esterified phytosterolson the one hand, causing conversion to a liquid crystalline state, andwith monoglycerides on the other hand to retard their crystallizationand induce a gel-like state. When combined in a ternary complex ofglycerine, phytoterols and monoglyceride, a highly dispersible productis created having both liquid crystalline and amorphous characteristics.

As described above, the addition of glycerine and its proposed complexformation with phytosterols has a very limited effect on thesolidification temperature and melting point of the phytosterols inspite of the fact that phytosterol crystallization is altered from hardcrystal to liquid crystal formation. This observation suggests that thethermodynamic stability of the liquid crystalline glycerine-phytosterolsolid complex is comparable to that of traditionally crystallizedphytosterol monohydrate. On the other hand, judging from the effect ofglycerine on the melting point of monoglycerides, glycerine causes agreater disruption of the crystalline structure and stability ofmonoglycerides. Thus, when 80% by weight of a monoglyceride-basedemulsifier (Myvatex 8-60, Kerry Ingredients, Beloit, Wis.) thatoriginally melts at a temperature of approximately 70° C. is mixed with20% by weight glycerine, the mixture melts and also begins tore-crystallize at approximately 68° C. As the proportion of glycerine isincreased to approximately 35% by weight, and the monoglyceride isdecreased to 65% by weight, crystallization and melting occur at asignificantly lower temperature, i.e., 62-63° C. rather than 70° C. Thissignificant 7-8° C. decrease in the monoglyceride melting temperaturesuggests both chemical dilution and de-stabilization of the crystallinemonoglyceride structure by glycerine. Moreover, while a 60%monoglyceride+40% glycerine melt is clear and fluid above 95-100° C., atlower temperatures (63°-95 C) a clear gel-like phase forms between theglycerine and the monoglyceride before crystallization commences at atemperature of 62-63° C. This glycerine-induced alteration ofmonoglyceride crystallization explains the observation that glycerinealso inhibits regular co-crystallization of monoglycerides withphytosterols when they are all mixed together and melted.

When a 1:1:1 mixture or a 0.5:1:1 mixture of glycerine, freephytosterols (FG-50 from Cargill, Inc., Minneapolis, Minn.), andmonoglycerides (e.g., Myvatex 8-60 containing approximately equalamounts of glyceryl monopalmitate and glyceryl monostearate) is heated,melted, and cooled, the glycerine substantially interferes with thephytosterols and monoglycerides forming conventional crystalline solids.Instead, a liquid crystalline complex is formed. As a beneficial result,the cooled liquid crystalline solids are readily dispersible in waterand aqueous beverages such as milk. Thus, glycerine not only produces anovel binary complex with phytosterols, but also forms a novel ternarycomplex that includes glycerine, phytosterols, and monoglyceridemolecules.

The ratio of glycerine, phytosterol, and monoglyceride components of aPGM ternary composition containing monoglycerides as the dispersingagent can vary depending on the relative amounts of phytosterol andmonoglyceride. The ratio of G to P, as for all phytosterol complexes ofthe invention, is in the range from about 0.05 g of G per gram of P toabout 1 gram of G to 1 gram of P. Similarly, the ratio of M to P is inthe range from about 0.1 to about 2.0, based on the weight of P. It isapparent, then, that the relative amounts of G and M can vary dependingon how much monoglyceride is used relative to the amount of phytosterol.In a preferred embodiment, the weight ratio of G:P:M is at least about0.5:1:1, where the amount of G can be 0.5 or greater and the amounts ofP and M are each 1. In another preferred embodiment, the weight ratio ofG:P:M is about 0.7:1:1. In another preferred embodiment, the weightratio of G:P:M is about 0.5:1:0.5. In another preferred embodiment, withthe weight of P held constant at 1 unit, the relative weights of G and Mcan be independently varied, each between approximately 0.4 and 1 unit.In yet another preferred embodiment, the weight ratio of G:M is about0.5 gram of G to about 1 gram of M. In still another preferredembodiment, the molar ratio of G:M is about 2:1.

Dispersal of PG or PGM microparticles in aqueous beverages and foods canbe further improved by including a small but effective amount of ionicsurfactant in the PGM blend. For example, about 1% to about 5% by weightof sodium stearate can be combined with, and added into anotheremulsifier. For example, 5% by weight sodium stearate may be combinedwith 95% by weight of a non-ionic emulsifier such as glycerylmonostearate and glyceryl monopalmitate. In particular with PGMmicroparticles, where glycerine already acts to prevent crystallizationof phytosterols and a dispersing agent such as a monoglyceride furtherpromotes dispersal, addition of an ionic surfactant produces a moreuniform emulsion. Better emulsions are expected to provide greatermicroparticle surface areas, and consequently better dispersal inbeverages such as milk and increased binding of both biliary anddietary-derived cholesterol in the gastrointestinal tract. Further, themonoglyceride component with its fatty acid binding to the phytosterolmolecule is expected to enhance cholesterol-phytosterol mixed micelleformation in the GI tract. All of these factors contribute to increasedphytosterol bioavailability, which is key to increasing the fecalelimination of cholesterol and decreasing the levels of plasmacholesterol.

The kinetics of the dispersal of PGM microparticles is rapid. Theirdispersal can be observed to occur rapidly and fully after mixing intoeither cold or hot aqueous beverages or foods, with the use of only lowto moderate shear force or agitation. Stable suspensions and emulsionscan be produced either by diluting and shear-mixing a semi-solid PGMconcentrate at ambient temperature in water, milk or other fluids, or byheat-softening or even re-melting the PGM complexes and then dispersingthem directly into a hot or cold aqueous medium. If desired, thesoftened or the melted composition can also be dispersed directly intoliquid, since the.re-melt temperatures of the ternary PGM complexes arebelow the boiling point of water. As yet another option for dispersal ofthe PGM material, after forming the PGM complex by melting, mixing andcooling the ingredients, an aqueous concentrate can be made by blending1 part of the PGM with, for example, 1-2 parts of milk, water or otheraqueous liquid. This concentrate can have the consistency of yogurt orsour cream, and can be easily dispersed in a beverage or food productusing low shear mixing. It can also be constituted as a gel, or gelledby rapid chilling. In either form, it can serve as an additive orcondiment to foods or beverages, such as coffee or tea.

Over a period of days following melt-blending of the PGM mixture, duringstorage at room temperature, the initially amorphous (non-crystalline)PGM complex may experience some growth of crystals containingphytosterol and monoglyceride. Since this is generally undesirable, andto assure that the PGM when added to a beverage or food is amorphous(with maximum microparticle surface area), the PGM can be remeltedbefore use. Remelting typically can be performed at 80-90° C. Afterremelting, it is stable for a period of hours to days.

For ease in dispersal, a PGM blend that has been gelled can also bepre-blended with a limited amount of cold or ambient temperature milk,water, or other liquid to form a concentrated aqueous pre-mix. Forexample, 1 part PGM gel can be mixed with 1-2 parts by weight of wateror milk to form a PGM concentrate or pre-mix. Approximately 3.0 g of theabove-described PGM blend containing equal parts by weight of freephytosterols, glycerine, and a monoglyceride such as Myvatex 8-60 willprovide a bioavailable daily level of more than 800 mg phytosterols asprescribed by the U.S. FDA for achieving a meaningful reduction inplasma cholesterol level and allowing a food product to carry theFDA-approved heart health claim for phytosterols.

Methods of Use

The compositions of the invention can be used to reduce the uptake ofsterols, such as cholesterol, in the gastrointestinal tract of a humanor animal subject, by including the compositions in solid or liquidfood, water, or nutritional or medical products ingested by the subject.

In one such method, a composition containing microparticulate PG or PGMcomplexes are administered to a subject in order to reduce one or moreplasma cholesterol levels in the subject. The subject consumes thephytosterol-glycerine complexes with food or drink, or as a supplementin the form of pills, capsules, powder, or liquid, and in the GI tractthe phystosterol binds and sequesters cholesterol present in the GItract and prevents its uptake into the bloodstream. It is well knownthat cholesterol can bind to phytosterols and reduce cholesterol uptake.Regular dietary intake of PG and PGM complexes, such as the intake ofapproximately 1-2 g of phytosterols per day, is known to typicallyresult in a substantial reduction in plasma LDL-cholesterol and totalcholesterol (see U.S. Pat. Nos. 7,709,038, 7,575,768, 7,144,595 and6,638,547. For a human subject, the subject preferably consumes a totalof about 0.5 g to about 4.0 g of phytosterols per day. The amountadministered can be adjusted in accordance with measurements of thesubjects cholesterol levels during the therapy, so as to obtain adesired range of cholesterol levels. A selected amount of PG or PGMcomplexes is consumed by the subject for a period of time sufficient toachieve the desired reduction in cholesterol levels. Preferably, PG orPGM is administered to the subject on a daily, alternating day, orweekly basis, and administration is continued for one or more weeks,months or years, or indefinitely, The amount of PG or PGM consumed bythe subject can also be varied depending on the anticipated amount ofcholesterol in the subjects daily diet. The amount of PG or PGMadministered to the subject can also be varied according to the actualor anticipated consumption of cholesterol on a meal-by-meal basis.

This method is capable of reducing both total plasma cholesterol andplasma LDL-cholesterol. In some embodiments of the method, the ratio ofLDL-cholesterol to HDL-cholesterol is reduced. The method can be used totreat hypercholesterolemia. The method also can be used to preventhypercholesterolemia by reducing plasma cholesterol levels in a subjectsuspected of having or acquiring hypercholesterolemia. In this context,prevention of hypercholesterolemia, or aiding in the prevention ofhypercholesterolemia, requires only that a reduction of plasmacholesterol levels is achieved in some subjects to whom it isadministered and, optionally, that the reduction is maintained withcontinued administration for a period of time, such as days, weeks,months, or years, so as to reduce the probability of the subjectacquiring hypercholesterolemia, or the symptoms resulting therefrom.

EXAMPLES Example 1 Formation of Phytosterol-Glycerine Complexes

Commercial crystalline phytosterol particles (CoroWise® FG-50 soybeanoil-derived phytosterols from the Cargill, Inc., Minneapolis, Minn.)were mixed with an excess of liquid glycerine on a microscope slide. Atambient temperature, little if any interaction between glycerine andcrystalline phytosterol particles was observed using phase contrastmicroscopy at 150× magnification. However, a physical transformation wasinduced by heating and melting the phytosterol particles suspended inglycerine to approximately 150° C. on a glass microscope slide using aBunsen burner and then allowing the slide to cool to room temperature.At the physical interface between crystalline phytosterol particles thathad melted, and the surrounding glycerine, the crystalline material wasreplaced by multiple layers of densely packed spherical phytosterolmicroparticles having a diameter of about 1 to 2 microns. Some of thismicroparticle material detached and became free-floating in theglycerine liquid, while the bulk of the microspherule material remainedbound to the bulk solid. Following cooling, the sample was examined bypolarized light microscopy. In a test tube, the microparticles could bereleased and dispersed into water using a vortex mixer. When viewed inpolarized light, most of the interior portions of the solidifiedmaterial consisted of radially “checkered” dark and light segmentedcircles of differing diameters; each segment appearing to have astriated and pleated surface. Upon rotation in the polarized light, boththe microparticles and the pleated, radially symmetric segmentsalternated between appearing bright and dark. It was concluded that theglycerine-complexed phytosterols contained liquid crystalline structure.

Example 2 Glycerine-Phytosterol Stoichiometry in Binary Compositions

An experiment was conducted to determine the “saturation uptake” levelduring the chemical interaction between phytosterols and glycerine, Thesaturation uptake level can be either an adsorption phenomenon or asolubility phenomenon. First, the saturation level of glycerine uptakeinto non-esterified phytosterols was determined Mixed phytosterols thathad been purified from soybean oil, and containing principallybeta-sitosterol, campesterol and stigmasterol (CoroWise® FG-50), wereheated, melted at a temperature of approximately 140-150° C. and cooledto room temperature. Increasing levels of glycerine (approximately 5,10, 20 and 25 parts by weight) could be adsorbed or dissolved in 100parts by weight of heated and melted free phytosterols. The evidence foradsorption (or solubility) was that a single melted phase of liquid wasvisible in heated glass test tubes containing these mixtures, whereaswhen larger amounts of glycerine were added, the glycerine formed aseparate clear phase beneath the melted phytosterols. Upon cooling andsolidification of the phytosterol samples containing betweenapproximately 5 and 25 parts by weight of glycerine, the resultingsolids were dry to the touch with no apparent free glycerine liquid.When 30, 40 or 50 parts by weight of glycerine were similarly dispersedand mixed with 100 parts by weight of the same heat-melted phytosterols,free glycerine liquid was evident as a separate phase beneath the moltenphytosterols in the heated test tubes, and the amount of free glycerineincreased as the weight fraction of glycerine was increased. Uponcooling of these samples, the uncomplexed glycerine was evident as anoily residue on the surface of the solids. Therefore, it was estimatedthat the saturation level for glycerine adsorbed or dissolved insoybean-derived free phytosterols (forming a liquid-solid solution) isbetween 20 and 30 parts by weight glycerine per 100 parts non-esterifiedphytosterols.

Example 3 Crystalline Versus Amorphous Phytosterol Morphologies

The chemical interaction between phytosterols and glycerine wasinvestigated using phase contrast and polarized light microscopy todistinguish crystalline from non-crystalline materials. Non-crystallineor “mesophase” materials are intermediate between crystalline andnon-crystalline phases. Samples of the above-described melted and cooledmixtures of glycerine (between 10 and 50 parts by weight) were combinedwith 100 parts phytosterols and were spread out on microscope slides toproduce thin coatings for microscopic examination. The first twosamples, containing 10 and 20 parts by weight glycerine, appearedentirely crystalline (i.e., they contained optically refracting, sharpedged plate-like crystals). However, the remaining samples containing30, 40 and 50 parts by weight glycerine per 100 parts free phytosterolswere unexpectedly converted to a material having a plastic consistencyand an amorphous (non-crystalline) oily appearance upon microscopicexamination. However, with transmitted polarized light examination, theamorphous-appearing material (both discrete microspheres and continuousregions) changed in appearance upon rotation of the sample on themicroscope stage, with different portions of the sample appearingalternately bright and dark. This behavior is diagnostic for liquidcrystalline material that, when fluid, still retains alignment ofmolecules in local regions.

It was found that 30 parts, but not 15 parts, by weight of glycerineconverted 100 parts by weight of free phytosterols from a crystallinestate to the amorphous, liquid crystalline state. In further experimentsit was determined that 25-30 parts by weight of glycerine also could becombined into heat-melted phytosterols, whereas 40-50 parts of glycerineclearly exceeded the solubility limit.

Example 4 Formation of Phytosterol-Glycerine-Monoglyceride Complexes

A combination of 0.5 parts by weight of food grade glycerine, 1.0 partby weight of vegetable oil-derived phytosterols (Cargill (Minneapolis,Minn.) FG-50 free phytosterols containing at least 90% by weightphytosterols), and 1.0 part by weight of the monoglyceride emulsifierMyvatex 8-60 (Kerry Food Ingredients, Inc., Beloit, Wis., containingapproximately 90% monoglycerides and 6% sodium stearate as a processingaid) was heated to approximately 100° C. (its melting point is 80-90°C., considerably less than that of the phytosterol alone (≧130° C.) andmelted together to form a homogeneous liquid mixture. The melt was thenallowed to cool to room temperature.

A similar experiment was carried out using a 1:1:1 (w/w) melt-blend ofpropylene glycol-phytosterol-Myvatex 8-60 (remelting temperature of85-87° C.). Doubling the amount of propylene glycol (2:1:1) onlyslightly decreased the re-melting temperature (83-85° C.) while makingthe cooled, solidified amorphous blend softer and somewhat easier tomanipulate.

Example 5 Dispersal of Phytosterol-Glycerine-Monoglyceride Complexes inBeverages and Foods

PGM complexes were made according to the method described in Example 4.The complexes were dispersed by blending directly in non-fat cow's milkand in regular soy milk Low to moderate shear force and/or agitation wasapplied using a Waring blender set at low to medium speed. By visualmonitoring, dispersal of the PGM complexes was seen to occur rapidly andcompletely after mixing into either cold or hot cow's milk or soymilk asdescribed above. The resulting suspensions and emulsions were stableover a period of at least one week when stored under conventionalrefrigeration at 4° C.

PGM complexes also were prepared using Myverol 18-04 (Kerry FoodIngredients, Inc.) as the emulsifier. Myverol 18-04 also containsglyceryl monostearate and monopalmitate, but without the 6% sodiumstearate processing agent found in Myvatex 8-60. The phytosterolcomplexes prepared with Myverol 18-04 were more difficult to disperseusing either vortex agitation or high shear Waring blending whencompared to the complexes prepared with Myvatex 8-60. Thus, PGMcomplexes containing a final level of approximately 2% sodium stearatewere more easily and effectively dispersed than similar PGM complexeswithout sodium stearate.

Example 6 Formation of Phytosterol-Monoglyceride Complexes

For comparison with the PGM complex, glycerine-free mixtures of 1:1(w/w) monoglyceride (Myvatex 8-60) and phytosterols were produced. Themonoglyceride and phytosterols were melted, cooled, and co-crystallizedto form “PM” complexes. While the PM complex, like the PG complex, iswater-dispersible, it disperses as generally crystalline particles thatare much larger, i.e., 20-100 microns in diameter, and therefore lessuseful in food and beverage formulations than the PGM microparticles.Such larger particles are expected to limit the ability of thephytosterol constituent to commingle with cholesterol at the molecularlevel in the digestive system, thereby reducing the efficacy ofphytosterol-promoted fecal elimination of cholesterol.

Example 7 Glycerine-Monoglyceride Stoichiometry in Ternary Compositions

Phytosterol-glycerine-monoglyceride ternary complexes were made asdescribed in Example 4. However the ratio of glycerine to monoglyceridewas varied according to the table below.

% Phytosterols Phytosterols Myvatex 8-60 Glycerine (wt/wt % Composition(weight) (weight) (weight) of total) A 1.00 1.00 0.70 37 B 1.00 0.750.58 43 C 1.00 0.50 0.45 51Each of the compositions A-C formed microparticles having liquidcrystalline structure and good to excellent dispersibility in water.Because composition C contains the highest proportion of phytosterols,it may be particularly useful in volume-limited applications such asdietary supplement gelatin capsules that are typically limited to anapproximate 1.0 g capacity in which approximately 0.5 g would bephytosterols.

Each of the foregoing patents, patent applications and references ishereby incorporated by reference.

What is claimed is:
 1. A method of reducing total plasma cholesterol(TC) level in a subject, the method comprising administering to thesubject an edible composition in an amount effective to reduce the TClevel in the subject, the edible composition comprising: (i) anon-esterified phytosterol, phytostanol, or a combination thereof(collectively “P”); and (ii) glycerine (“G”); wherein P and G arecommingled in the composition to form, at least in part, a PG molecularcomplex; and wherein the weight ratio of G:P in the composition is atleast 0.05:1.
 2. The method of claim 1, wherein the subject is human andfrom about 0.5 g to about 4.0 g of non-esterified phytosterols isingested daily by the human subject.
 3. The method of claim 2, whereinthe human subject consumes 1-2 g per day of non-esterified phytosterols.4. The method of claim 1, wherein the edible composition is consumed ona daily, alternating day, or weekly basis.
 5. The method of claim 1,wherein the administration of the edible composition to the subjectcontinues for one or more weeks, months, or years.
 6. The method ofclaim 1, wherein the edible composition is a beverage, food product,nutritional supplement, dietary supplement, or liquid food additive. 7.The method of claim 1, wherein the edible composition is consumedtogether with food or drink.
 8. The method of claim 1, wherein theedible composition is in the form of pills, capsules, powder, or liquid.9. The method of claim 1, wherein the level of LDL-cholesterol in thesubject is also reduced.
 10. The method of claim 1, wherein the ratio ofLDL-cholesterol to HDL-cholesterol in the subject is also reduced. 11.The method of claim 1, wherein the composition is administered in anamount effective to bind cholesterol in the gastrointestinal tract ofthe subject and prevent or reduce its uptake.
 12. The method of claim 1,wherein the subject is suspected of having or acquiringhypercholesterolemia.
 13. The method of claim 1 that is used to treat orprevent hypercholesterolemia.
 14. The method of claim 1, wherein P isselected from the group consisting of beta-sitosterol, beta-sitostanol,campesterol, campestanol, stigmasterol, stigmastanol, brassicasterol,brassicastanol, clionasterol and clionastanol, and combinations thereof.15. The method of claim 1, wherein P is hydrated, hemi-hydrated,dehydrated or a combination thereof.
 16. The method of claim 1, whereinthe molar ratio of G:P is about 1:1 or greater.
 17. The method of claim1, wherein the commingled P and G are in the form of microparticleshaving a diameter of less than 5 microns.
 18. The method of claim 17,wherein the microparticles have a diameter of about 2 microns or less.19. The method of claim 1, wherein at least a portion of said PGmolecular complex is present in a liquid crystalline form.
 20. Themethod of claim 1, wherein the composition further comprises adispersing agent (“M”), and wherein the weight ratio of M:P is fromabout 0.1:1 to about 2:1.
 21. The method of claim 20, wherein the weightratio of M:P is from about 0.3:1 to about 1:1.
 22. The method of claim20, wherein M is a monoglyceride, a diglyceride, a lecithin, an ionicsurfactant, or a combination thereof.
 23. The method of claim 22,wherein M comprises a monoglyceride selected from the group consistingof glyceryl monostearate, glyceryl monopalmitate, and combinationsthereof.
 24. The method of claim 22, wherein M comprises an ionicsurfactant present in a concentration from about 1% to about 10% byweight based on the total amount of M.
 25. The method of claim 22,wherein M is an ionic surfactant which is a salt of a fatty acid,wherein the fatty acid is selected from the group consisting of stearicacid, palmitic acid, myristic acid, lauric acid, capric acid, caprylicacid, oleic acid, and combinations thereof.
 26. The method of claim 22,wherein M comprises a monoglyceride and 5% sodium stearate.
 27. Themethod of claim 22, wherein M comprises hydrolyzed lecithin and an ionicsurfactant.
 28. The method of claim 22, wherein the ionic surfactant issodium stearate.
 29. The method of claim 22, wherein M comprises alecithin selected from the group consisting of hydroxylated lecithin andhydrolyzed lecithin.